Analytical and Clinical Validation for RT-qPCR Detection of SARS-CoV-2 Without RNA Extraction.

José P Miranda,Rossana Camponovo,Gladys Angel,Javiera Osorio,Marcela Henríquez-Henríquez,Mauricio Videla

doi:10.3389/fmed.2020.567572

Abstract

Background: The recent COVID-19 pandemic has posed an unprecedented challenge to laboratory diagnosis, based on the amplification of SARS-CoV-2 RNA. With global contagion figures exceeding 4 million persons, the shortage of reagents for RNA extraction represents a bottleneck for testing globally. We present the validation results for an RT-qPCR protocol without prior RNA extraction. Due to its simplicity, this protocol is suitable for widespread application in resource-limited settings.Methods: Optimal direct protocol was selected by comparing RT-qPCR performance under a set of thermal (65, 70, and 95° for 5, 10, and 30 min) and amplification conditions (3 or 3.5 uL loading volume; 2 commercial RT-qPCR kits with a limit of detection below 10 copies/reaction) in nasopharyngeal swabs stored at 4°C in sterile Weise's buffer pH 7.2. The selected protocol was evaluated for classification concordance with a standard protocol (automated RNA extraction) in 130 routine samples and 50 historical samples with Cq values near to the clinical decision limit.Results: Optimal selected conditions for direct protocol were: thermal shock at 70°C for 10 min, loading 3.5 ul in the RT-qPCR. Prospective evaluation in 130 routine samples showed a 100% classification concordance with the standard protocol. The evaluation in historical samples, selected because their Cqs were at the clinical decision limit, showed 94% concordance with our confirmatory standard, which includes manual RNA extraction.Conclusions : Our results validate the use of this direct RT-qPCR protocol as a safe alternative for SARS-CoV-2 diagnosis in the case of a shortage of reagents for RNA extraction, with minimal clinical impact.

Highlights

In late 2002, an epidemic outbreak of severe acute respiratory syndrome (SARS) was described in China’s Guangdong province, and its cause was attributed months later to the SARS-CoV coronavirus [1,2,3]
Until May 9, 2020, the Foundation for Innovation in New Diagnostics (FIND; http://www.finddx.org) listed on its website at least 141 different commercially available diagnostic kits for nucleic acid amplification tests (NAAT) for SARS-CoV-2 with CEIVD certification, and this number increased to 314 kits when considering other types of certification for clinical diagnosis
Using the direct protocol saved about 40% of the analysis time compared to the standard, which allowed for enhanced daily processing capability

Summary

Introduction

In late 2002, an epidemic outbreak of severe acute respiratory syndrome (SARS) was described in China’s Guangdong province, and its cause was attributed months later to the SARS-CoV coronavirus [1,2,3]. A new strain of coronavirus (SARS-CoV-2), causing COVID19 disease, was reported in December 2019 in Wuhan, Hubei province, China [5]. Since this outbreak has spread globally, forcing the WHO to decree a pandemic on March 11, 2020, when the number of confirmed cases reached 118,000 within 114 countries [6]. Until May 9, 2020, the Foundation for Innovation in New Diagnostics (FIND; http://www.finddx.org) listed on its website at least 141 different commercially available diagnostic kits for nucleic acid amplification tests (NAAT) for SARS-CoV-2 with CEIVD certification, and this number increased to 314 kits when considering other types of certification for clinical diagnosis. This protocol is suitable for widespread application in resource-limited settings

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